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Originally published In Press as doi:10.1074/jbc.M702344200 on November 8, 2007

J. Biol. Chem., Vol. 283, Issue 3, 1350-1361, January 18, 2008
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Stimulatory Heterotrimeric GTP-binding Protein Inhibits Hydrogen Peroxide-induced Apoptosis by Repressing BAK Induction in SH-SY5Y Human Neuroblastoma Cells*

So-Young Kim{ddagger}, MiRan Seo{ddagger}, Yeni Kim{ddagger}, Yun-Il Lee{ddagger}, Jung-Min Oh{ddagger}, Eun-Ah Cho{ddagger}, Jae-Seung Kang§, and Yong-Sung Juhnn{ddagger}1

From the {ddagger}Department of Biochemistry and Molecular Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799 and the §Department of Microbiology, College of Medicine, Inha University, Incheon 402-751, Korea

Received for publication, March 19, 2007 , and in revised form, October 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterotrimeric stimulatory GTP-binding protein (Gs) stimulates adenylate cyclases to activate the cAMP signaling pathway. Although the cAMP pathway has been reported to be involved in apoptosis, the role of the Gs-cAMP signaling pathway during reactive oxygen species (ROS)-mediated apoptosis, which is involved in the resistance of cancer cells to chemotherapy and radiation, is not clearly understood. Thus, in this study we aimed to investigate the role of the {alpha} subunit of Gs (G{alpha}s) in the ROS-induced apoptosis of cancer cells. The stable expression of constitutively active G{alpha}s (G{alpha}sQL) inhibited the hydrogen peroxide-induced apoptosis of SH-SY5Y human neuroblastoma cells and reduced the hydrogen peroxide-induced increase in Bak and the decrease in Bcl-xL protein expression. Exogenous Bak expression abolished these inhibitory effects of G{alpha}sQL, but Bak small interfering RNA decreased hydrogen peroxide-induced apoptosis. G{alpha}s repressed hydrogen peroxide-induced Bak expression by inhibiting the transcription of Bak mRNA, which resulted from the inhibition of the hydrogen peroxide-induced activation of transcription factors such as AP1, NF-{kappa}B, and NFAT. Moreover, G{alpha}s also inhibited the hydrogen peroxide-induced binding of AP1, NF-{kappa}B, and NFAT to the Bak promoter. Furthermore, hydrogen peroxide-induced apoptosis was reduced by treating cells with prostaglandin E2, which activates G{alpha}s, but this was augmented by CCPA, which activates G{alpha}i causing a decrease in cAMP levels. From the results, we conclude that G{alpha}s protects neuroblastoma cells from hydrogen peroxide-induced apoptosis by repressing Bak induction, which is mediated by the inhibition of the hydrogen peroxide-induced activations of AP1, NF-{kappa}B, and NFAT through cAMP-PKA-CREB signaling system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The heterotrimeric GTP-binding proteins (G proteins)2 are composed of {alpha}, β, and {gamma} subunits, and the {alpha} subunits of G protein (G{alpha}) are classified into four main families: G{alpha}s, G{alpha}i, G{alpha}q, and G{alpha}12. When a signaling molecule, such as a hormone or a neurotransmitter binds to a G protein-coupled receptor (GPCR), the receptor stimulates the replacement of GDP with GTP on the G{alpha}. GTP-bound activated heterotrimer dissociates into an {alpha} subunit (G{alpha}-GTP) and a β{gamma} dimer (Gβ{gamma}), which can independently regulate effectors including adenylate cyclases, phospholipases, phosphodiesterases, and ion channels. The hydrolysis of GTP to GDP by intrinsic GTPase, a process that is regulated by RGS (regulator of G-protein signaling) proteins, leads to the reassociation of the heterotrimer and the termination of the activation cycle (1–3). G protein signaling systems are involved in regulation of a variety of cellular responses, which include metabolism, neurotransmission, proliferation, differentiation, and apoptosis. Although signaling for cellular proliferation and apoptosis has generally been attributed to growth factor receptors that possess ligand-regulated protein-tyrosine kinase activity, growing evidence now indicates that GPCRs also regulate cellular growth and apoptosis (4–6).

Reactive oxygen species (ROS) are constantly generated under normal conditions as a consequence of aerobic metabolism, and the most common ROS types are superoxide anions (Formula), hydrogen peroxide (H2 O2), and hydroxyl radicals (HO–) (7). ROS can react with DNA, proteins, carbohydrates, and lipids in a destructive manner due to their high levels of chemical reactivity. Thus, ROS are considered DNA-damaging agents that increase mutation rates and promote oncogenic transformation, and are implicated in the development of cancer and metastases (8, 9). Moreover, ROS participate in the modulation of apoptosis following treatment with various agents including Fas, ultraviolet, and chemotherapeutic drugs (10–12). Therefore, the ability of a cancer cell to defend itself against ROS is associated with resistance to chemo- and radiotherapy. Consequently, a more detailed understanding of the mechanisms that regulate ROS-induced apoptosis would contribute to our abilities to develop novel therapeutic strategies directed toward killing resistant cancer cells (13).

The {alpha} subunit of the stimulatory G protein (G{alpha}s) activates adenylate cyclases to increase cellular cAMP, which regulates various cellular responses by activating cAMP-dependent protein kinase (PKA), guanine nucleotide exchange factor activated by cAMP, and cyclic nucleotide gated ion channels. Moreover, increased G{alpha}s signaling was found to lead to the formation of tumors in endocrine cells (14), and thus, the activated mutant of G{alpha}s is known as gsp oncogene. Increased G{alpha}s signaling also leads to the apoptosis of cardiac myocytes (4) but protects neuronal apoptosis (15). However, the effect of G{alpha}s signaling on ROS-mediated death of cancer cells is not understood. Thus, we undertook this study to examine whether G{alpha}s can modulate hydrogen peroxide-induced cancer cell death, and if so, to elucidate the molecular mechanism responsible.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents—SH-SY5Y human neuroblastoma cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA), and were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (JBI, Korea) and 100 units/ml penicillin/streptomycin, in a5%CO2 incubator at 37 °C. SH-SY5Y cells stably expressing constitutively active mutant G{alpha}s (G{alpha}sQ227L) were established previously as described (16). Hydrogen peroxide, 2-chloro-N(6)-cyclopentyl-adenosine (CCPA), cycloheximide, LiCl, pyrrolidine dithiocarbamate, prostaglandin E2 (PGE2), and SB203580 were purchased from Sigma, cyclosporine A from Tocris (Bristol, UK), and H89 and forskolin from Calbiochem (La Jolla, CA).

Expression Constructs and Transient Transfection—cDNA encoding constitutively active G{alpha}i2 (G{alpha}i2QL) was subcloned into pcDNA3 expression vector (Invitrogen). The Bak-pcDNA3 expression construct was kindly provided by Dr. Young-Jun Oh (Yonsei University, Korea). Reporter plasmids containing luciferase gene under the control of CRE, AP1, NFAT, and NF-{kappa}B, respectively, were from Stratagene (La Jolla, CA). Decoy oligonucleotides for the cAMP response element (decoy CRE) were constructed of phosphorothioate oligonucleotide (Genotech, Korea), with the following sequences 5'-TGACGTCATGACGTCATGACGTCA-3' (24 bp), and the sequences of control decoy were 5'-CTAGCTAGCTAGCTAGCTAGCTAG-3' (24 bp). pENTR/H1/TO-G{alpha}s and the Bak siRNA construct were generated by the BLOCK-iTTM Inducible H1 RNAi Entry Vector Kit (Invitrogen) using the following primers: G{alpha}s siRNA, a forward primer 5'-CACCGCGATGTGACTGCCATCATCCGAAGATGATGGCAGTCACATCG-3' and a reverse primer 5'-AAAACGATGTGACTGCCATCATCTTCGGATGATGGCAGTCACATCGC-3'; Bak siRNA, a forward primer 5'-CACCGAACCGACGCTATGACTCAGAGCGAACTCTGAGTCATAGCGTCGGTT-3' and a reverse primer 5'-AAAAAACCGACGCTATGACTCAGAGTTCGCTCTGAGTCATAGCGTCGGTTC-3'. Transfection was performed by electroporation using a Gene Pulser II (Bio-Rad) at 250 V/950 microfarads.

Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)—Total RNA was isolated from the cells using the acid guanidinium thiocyanate-phenol-chloroform ex-traction method (17). First-strand complementary DNA (cDNA) was synthesized using oligo(dT) primers and the SuperScript First-Strand Synthesis System (Invitrogen). PCR was performed with specific primers: Bak, forward primer 5'-TGAAAAATGGCTTCGGGGCAAGGC-3' and reverse primer 5'-TCATGATTTGAAGAATCTTCGTACC-3'; glyceraldehyde-3-phosphate dehydrogenase, forward primer 5'-ACCACAGTCCATGCCATCAC-3' and reverse primer 5'-TCCACCACCCTGTTGCTGTA-3'. Real-time RT-PCR was performed in a 25-µl mixture composed of 200 nM forward and reverse primers, and iQTM SYBR Green Supermix (Roche, Basel, Switzerland) using iCyler and iQ software (Bio-Rad). After 40 cycles of PCR, average Bak threshold cycle (Ct) values from triplicate PCR were normalized against the average Ct values of glyceraldehyde-3-phosphate dehydrogenase.

Immunoblot Analysis—Immunoblotting was performed as previously described (18). Total cell lysates (50 µg of protein) were separated on 10 or 15% SDS-polyacrylamide gels, transferred to nitrocellulose paper, and analyzed with specific antibodies. Antibodies against Bcl2, G{alpha}i2, GSK-3β, I{kappa}B{alpha}, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA), antibodies against Bcl-xL, Bad, Bax, Bak, phosphorylated CREB (Ser133), cleaved caspase-3 (Asp175), c-Fos, phosphorylated GSK-3β (Ser9), poly(ADP-ribose) polymerase (PARP), JNK, phosphorylated JNK (Thr183/Tyr185), and phosphorylated c-Jun (Ser63, Ser73) were from Cell Signaling Technology (Beverly, MA). Antibody against NFAT1 was purchased from Affinity Bioreagents (Golden, CO). Cytochrome c released into the cytoplasm was analyzed after subcellular fractionation by immunoblotting using an antibody obtained from BD Biosciences (19). The densities of visualized bands were quantified using NIH Image J software and relative band densities are expressed as percentages of corresponding control densities.

Flow Cytometric Analysis of Annexin V and Propidium Iodide-stained Cells—Apoptosis was quantified using Annexin V-FITC apoptosis kits (BD Biosciences) according to the manufacturer's instructions. Stained cells were analyzed using a FACSCalibur flow cytometer and the CellQuest analysis program (BD Biosciences NorthRyde, Australia).

Generation of Bak-Promoter Reporter Constructs—Genomic DNA was isolated from SH-SY5Y cells by digestion with proteinase K and sequential phenol extraction (20), and used as a template for PCR to clone the Bak promoter. The forward primer designed for cloning the Bak promoter region contained an XhoI site at the 5' terminus, with the sequence 5'-CCGCTCGAGTGCACAGGTCTAAATTGCAGG-3'. The reverse primer contained a HindIII site at the 5' terminus, and had the sequence 5'-CCCAAGCTTGCACTCATGGTTATGGGATGG-3'. PCR was performed using LA Taq polymerase (Takara Shuzo Co., Japan), and the resulting 3500-bp PCR products were digested with XhoI and HindIII and then ligated upstream of the luciferase reporter gene in pGL2 basic vector (Promega Corp., Madison, WI) to generate Bak-pLuc. A series of mutants with various deletions in the cloned Bak promoter region were prepared by PCR. The primers used were as follows: for mutant deleted between –3500 and –2500, (–2500)Bak-pLuc: a forward primer 5'-CCGCTCGAGGGTCTTGTCCATCCTCCCAGA-3'; for mutant deleted between –3500 and –1500, (–1500)Bak-pLuc: a forward primer 5'-CCGCTCGAGCTTCACCCTGTCCCTGTCCGG-3'; and for mutant deleted between –3500 and –522, (–522)Bak-pLuc: a forward primer 5'-CCGCTCGAGGGAACAGCTAAAAACCCCCCA-3'. Mutants deleted in AP1 sites in Bak promoter of (–1500)Bak-pLuc were also constructed by PCR using the following primers: D1 (deletion upstream of –1216), 5'-CCGCTCGAGGTGACTAACGATGTGACCTCG-3'; D2 (deletion between –912 and –919) products were constructed by ligating two PCR products into wild type plasmid digested with XhoI and HindIII (XhoI-SacII fragment: forward primer 5'-CCGCTCGAGCTTCACCCTGTCCCTGTCCGG-3', reverse primer 5'-TCCCCGCGGAACCTGCTGTGACCCCTCATC-3', SacII-XhoI fragment: forward primer 5'-TCCCCGCGGACCTCGGGCACTCAACTCTTT-3', reverse primer 5'-CCCAAGCTTGCACTCATGGTTATGGGATGG-3'; D1 and D2 (deletion upstream of –912), 5'-CCGCTCGAGCGATGTGACCTCGGGCACTCA-3'. The same reverse primer (5'-CCCAAGCTTGCACTCATGGTTATGGGATGG-3') was used all PCRs for mutagenesis.

Luciferase Activity Assays—SH-SY5Y cells were transfected with plasmids containing luciferase reporter gene by electroporation. Luciferase activities were assayed using the Bioluminescent Reporter Gene Assay System (Tropix, Bedford, MA) according to the manufacturer's instructions. At least three independent experiments in duplicate were performed, and promoter activities were normalized versus β-galactosidase activity.

Electrophoretic Mobility Shift Assay by Chemiluminescence—The cells were treated with hydrogen peroxide for 24 h, and then disrupted in lysis buffer (containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40). The cell lysates was centrifuged at 14,000 x g at 4°C for 6 min, and the resulting pellets were resuspended in extraction buffer (20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 25% glycerol, 0.2 mM EDTA, and 1% Nonidet P-40). After centrifugation, the supernatant was collected for study of DNA-protein interactions, which were analyzed by chemiluminescence using the LightShift Chemiluminescent EMSA Kit (Pierce). Oligonucleotides containing AP1, NFAT, NF-{kappa}B, or CREB binding site sequences in the Bak promoter region were 5' end-labeled with biotin. Sequences were as follows: AP1 binding site, 5'-CTTTCCGTGACTAACGATGTGACC-3'; CREB binding site, 5'-GAGTGCAGTGACGTCATCTCGGCT-3'; NFAT binding site, 5'-GGCAAAGAGGAAAATTTGGGGCCG-3'; and NF-{kappa}B binding site, 5'-GAAGTTGAGGGGGACTTTCCCAGG-3'.

Measurement of cAMP Accumulation—cAMP contents were determined by measuring the competitive binding of [3H]cAMP to a cAMP-binding protein, the regulatory subunit RI{alpha} of the cAMP-dependent protein kinase was expressed in Escherichia coli. In brief, the culture medium was removed from a 12-well plate containing the SH-SY5Y neuroblastoma cells, and cAMP was extracted by immediate addition of 2.0 M perchloric acid. After the acid extract was neutralized with 4.0 M KOH, cAMP levels were determined and normalized to the amount of acid-insoluble protein.

Data Analysis—At least three independent experiments were conducted in all cases. Data are presented as mean ± S.E. The nonparametric Mann-Whitney U test was used to analyze mean values, and p values < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
G{alpha}s Inhibited Hydrogen Peroxide-induced Apoptosis of SH-SY5Y Neuroblastoma Cells—To investigate the role of G{alpha}s in hydrogen peroxide-induced apoptosis, a constitutively active mutant G{alpha}s, G{alpha}sQ227L (G{alpha}sQL), was stably expressed in SH-SY5Y human neuroblastoma cells; this was confirmed by Western blotting using antibodies against G{alpha}s and hemagglutinin tag in G{alpha}sQL (Fig. 1A). The expression of G{alpha}sQL increased the basal cAMP level to 28.5 ± 0.5 pmol/mg protein (p < 0.05) from 3.4 ± 0.6 pmol/mg of protein in vector-transfected cells, and the CREB phosphorylation. Twenty-four hours after treatment with 100 µM hydrogen peroxide for 30 min, cells were stained with Annexin V to detect apoptosis. Stable expression of G{alpha}sQL reduced Annexin V positive and propidium iodide-negative cells to 9.6 ± 1.7 from 45.8 ± 4.5% (p < 0.02), and Annexin V-positive and propidium iodide-positive cells to 3.9 ± 1.1 from 15.0 ± 2.2% (p < 0.02) in vector-transfected control (Fig. 1B). G{alpha}sQL expression also decreased the cleavages of caspase-3 and PARP (Fig. 1C), and blocked cytochrome c release into cytosol (Fig. 2A) after 24 h of hydrogen peroxide treatment. To confirm the anti-apoptotic effect of G{alpha}s, the effects of G{alpha}s siRNA were analyzed. The transient transfection of G{alpha}s siRNA decreased the basal expression of G{alpha}s in the G{alpha}sQL expressing cells (56.6 ± 2.9%, p < 0.05) and vector-transfected cells (46.0 ± 4.5%, p < 0.05) from siRNA-untransfected controls. Treatment with hydrogen peroxide augmented the siRNA effect resulting in a further decrease of G{alpha}s expression in the G{alpha}sQL expressing cells (40.2 ± 1.4%, p < 0.05) and vector-transfected cells (26.2 ± 4.7%, p < 0.05) (Fig. 1D). Transfection of G{alpha}s siRNA increased hydrogen peroxide-induced caspase-3 cleavage to 193.3 ± 2.3% (p < 0.05) and PARP to 146.3 ± 4.4% (p < 0.05) in vector-transfected cells, and it also increased hydrogen peroxide-induced caspase-3 cleavage from 36.6 ± 4.9 to 162.6 ± 4.6% (p < 0.05) and PARP from 24.0 ± 2.7 to 129.8 ± 4.7% (p < 0.05) in G{alpha}sQL expressing cells versus the hydrogen peroxide-treated vector-transfected control (Fig. 1D), indicating that G{alpha}s protected SH-SY5Y neuroblastoma cells from hydrogen peroxide-induced apoptosis.

G{alpha}s Inhibited Hydrogen Peroxide-induced Apoptosis by Repressing Increases in Bak Protein in SH-SY5Y Neuroblastoma Cells—To probe the mechanism whereby G{alpha}s inhibits hydrogen peroxide-induced apoptosis of the neuroblastoma cells, we examined the effects of G{alpha}s on the expression of Bcl2 family proteins. Treatment with hydrogen peroxide induced an increase in pro-apoptotic Bak protein, but decreases in Bax and Bad proteins. Moreover, the stable expression of G{alpha}sQL inhibited up-regulation of Bak and down-regulation of Bax and Bad induced by hydrogen peroxide (Fig. 2A). However, hydrogen peroxide treatment did not decrease anti-apoptotic Bcl-expression, and the overexpression of G{alpha}sQL did not change Bcl2 expression. The expression of Bcl-xL, another anti-apoptotic Bcl2 family protein, was reduced by hydrogen peroxide treatment, and the overexpression of G{alpha}sQL blocked the hydrogen peroxide-induced reduction in Bcl-xL expression (Fig. 2A). Moreover, treatment with hydrogen peroxide increased mitochondrial Bak protein to 3.1 ± 0.3-fold (p < 0.02) in vector-transfected cells. The stable expression of G{alpha}sQL resulted in an increase in the basal level of mitochondrial Bak by 1.5 ± 0.2-fold (p < 0.02), but reduced the hydrogen peroxide-induced expression of Bak to 1.7 ± 0.1-fold (p < 0.02) of the untreated vector control level (Fig. 2B).


Figure 1
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  		FIGURE 1.
G{alpha}s inhibited hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells. A, stable expression of hemagglutinin-tagged G{alpha}sQL. M represents mock-control cells, V represents vector-transfected cells, and the number represents the clone number. B and C, inhibition of hydrogen peroxide-induced apoptosis by G{alpha}sQL, as assessed by FACS analysis of Annexin V and propidium iodide (PI)-stained cells (B), and Western analysis of PARP and caspase-3 cleavage (C). SH-SY5Y cells were treated with 100 µM hydrogen peroxide for 24 h, and then apoptosis was assessed by FACS analysis and Western blotting. The top panel shows representative FACS analysis data, whereas histograms represent mean ± S.E. V represents vector-transfected cells and G represents G{alpha}sQL-transfected cells. Asterisks indicate significant difference from vector-transfected controls (p < 0.05, Mann-Whitney U test). D, G{alpha}s knock-down reduced the anti-apoptotic effects of G{alpha}s. SH-SY5Y cells were transfected with 30 µg of pENTR-G{alpha}s siRNA or pENTR-lacZ vector by electroporation, and cells were treated with 100 µM hydrogen peroxide at 48 h after transfection. Then apoptosis was assessed by analysis of the cleavages of caspase-3 and PARP by Western blotting at 24 h.

 
To confirm the participation of Bak in the anti-apoptotic effect of G{alpha}s, the effects of the transient expressions of Bak and Bak siRNA were analyzed. Overexpression of Bak caused an increase in the hydrogen peroxide-induced cleavages of PARP and caspase-3 in both vector-transfected and G{alpha}sQL-transfected neuroblastoma cells (Fig. 2C). On the other hand, the expression of Bak siRNA decreased basal and hydrogen peroxide-induced Bak expressions in both G{alpha}sQL expressing cells and vector-transfected cells, and inhibited hydrogen peroxide-induced caspase-3 cleavage to 61.6 ± 17.4% (p < 0.02) and PARP cleavage to 51.8 ± 15.8% (p < 0.02) of the vector transfected control level (Fig. 2D).

G{alpha}s Repressed Hydrogen Peroxide-induced Bak Expression by Inhibiting the Transcription of Bak mRNA—To investigate the mechanism whereby G{alpha}s represses the hydrogen peroxide-induced expression of Bak protein, we examined the degradation rate of Bak protein after blocking protein synthesis with cycloheximide. It was found that the degradation rate of Bak protein in G{alpha}sQL expressing cells was not significantly higher than that in vector-transfected cells (Fig. 3A). On the other hand, hydrogen peroxide treatment markedly increased Bak mRNA in vector-transfected cells, but only slightly in G{alpha}sQL-transfected cells (Fig. 3B). This finding was confirmed by real time RT-PCR, which showed a 29.0 ± 4.5-fold (p < 0.02) increase in Bak mRNA in hydrogen peroxide-treated vector-transfected control cells, and a 6.5 ± 2.3-fold increase (p < 0.02) in G{alpha}sQL expressing cells versus the untreated vector-transfected control cells. In addition, expression of G{alpha}sQL increased the basal Bak mRNA level to 3.2 ± 0.7-fold (p < 0.02) versus the untreated vector-transfected control cells (Fig. 3C). Furthermore, hydrogen peroxide treatment increased luciferase activity under the control of the Bak promoter to 12.6 ± 0.3-fold (p < 0.02) compared with untreated vector-transfected cells. The expression of G{alpha}sQL increased basal Bak luciferase activity to 1.8 ± 0.5-fold (p < 0.02), but reduced hydrogen peroxide-stimulated luciferase activity to 3.1 ± 0.5-fold of the untreated vector-transfected cells (p < 0.05).

G{alpha}s Inhibited the Transcription of Bak mRNA via cAMP-PKA-CREB-dependent Pathways—In a study to probe the signaling pathway mediating the repression of Bak induction by G{alpha}s, we treated SH-SY5Y neuroblastoma cells with forskolin. Forskolin treatment reduced the hydrogen peroxide-induced cleavages of PARP and caspase-3 and the release of cytochrome c to the cytosol (Fig. 4A). Similar protective effects were exhibited by treatment of the cells with dibutyryl cAMP (data not shown). Moreover, forskolin treatment blocked the increase in Bak and the decrease in Bad and Bax induced by hydrogen peroxide. Forskolin also reduced the observed down-regulation of Bcl-xL by hydrogen peroxide, but did not change Bcl2 expression (Fig. 4A). Treatment of cells with H89, a cAMP dependent protein kinase (PKA) inhibitor, abolished the anti-apoptotic effects of G{alpha}sQL and the inhibitory effect of G{alpha}sQL on Bak induction, as evidenced by PARP and caspase-3 cleavage (Fig. 4B). Furthermore, transfection of decoy CRE abolished the anti-apoptotic effects of G{alpha}sQL and its inhibition of Bak induction by hydrogen peroxide (Fig. 4C).


Figure 2
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  		FIGURE 2.
G{alpha}s inhibited apoptosis by repressing Bak expression induced by hydrogen peroxide. A, effects of G{alpha}sQL on the hydrogen peroxide-induced expressions of Bcl2 family proteins. B, effects of G{alpha}sQL on the hydrogen peroxide-induced expression of Bak. After incubation with 100 µM hydrogen peroxide for 24 h, cells were analyzed for Bcl2 family proteins and the mitochondrial distribution of Bak. Histograms represent mean ± S.E. and Bak densities expressed as -fold of the corresponding densities of control cells. Asterisks indicate significantly different versus vector-transfected cells (p < 0.05, Mann-Whitney U test). The effects of Bak overexpression (C) and Bak knockdown (D) on the anti-apoptotic effects of G{alpha}s. SH-SY5Y cells were transfected with 15 µg of Bak plasmids (pcDNA3-Bak or pENTR-Bak siRNA) or vector controls (pcDNA3 or pENTR-lacZ) by electroporation. After 48 h, cells were then treated with 100 µM hydrogen peroxide for 24 h and apoptotic levels were determined.

 
G{alpha}s Repressed Hydrogen Peroxide-induced Bak Expression by Inhibiting the Activations of Transcription Factors—To investigate the mechanism underlying the repression of hydrogen peroxide-induced Bak transcription by G{alpha}s, the effects of G{alpha}sQL on the activation of transcription factors by hydrogen peroxide were analyzed. Treatment with hydrogen peroxide activates reporter luciferase under the control of transcription factors AP1, NFAT, and NF-{kappa}B, but not under the control of CREB. The expression of G{alpha}sQL activated CREB-luciferase activity to 3.5 ± 0.4-fold (p < 0.02) of the basal activity, but inhibited the hydrogen peroxide-induced activation of AP1-luciferase from 11.5 ± 1.2- to 3.8 ± 0.4-fold (p < 0.02), NFAT-luciferase from 4.6 ± 0.8- to 1.6 ± 0.1-fold (p < 0.02), and NF-{kappa}B-luciferase from 5.2 ± 0.6- to 1.6 ± 0.5-fold (p < 0.02) (Fig. 5A). Similarly, treatment with forskolin activated CREB-luciferase activity to 11.4 ± 0.7-fold (p < 0.02) of basal activity, but inhibited hydrogen peroxide-induced activation of AP1-luciferase from 9.7 ± 0.2- to 1.9 ± 0.2-fold (p < 0.02), NFAT-luciferase from 3.7 ± 0.3- to 1.2 ± 0.4-fold (p < 0.02), and NF-{kappa}B-luciferase from 4.2 ± 0.1- to 1.0 ± 0.3-fold (p < 0.02) (Fig. 5A). Furthermore, hydrogen peroxide-induced Bak-luciferase activities were inhibited by pre-treatment with SB203580 (an AP1 inhibitor), cyclosporine A (an NFAT inhibitor), or pyrrolidine dithiocarbamate (an NF-{kappa}B inhibitor) to 2.1 ± 1.1-, 4.3 ± 0.4-, and 3.3 ± 1.0-fold, respectively, from 13.6 ± 0.2-fold of the control (p < 0.02), but pre-treatment with CRE decoy enhanced hydrogen peroxide-induced Bak-luciferase activity to 29.8 ± 0.5-fold from the 13.6 ± 0.2-fold of the control (p < 0.02) (Fig. 5B).

G{alpha}s Repressed Hydrogen Peroxide-induced Bak Expression by Inhibiting the Binding of Transcription Factors on Bak Promoter—To localize the region of the Bak promoter responsible for hydrogen peroxide induction, the effects of serial deletion on Bak luciferase activity were analyzed in SH-SY5Y neuroblastoma cells. Treatment with hydrogen peroxide induced Bak luciferase activity to 12.9 ± 0.2-fold under the control of the –3500-bp untranslated region, to 9.5 ± 0.7-fold under the control of –2500-bp untranslated region, and 6.1 ± 0.1-fold under the control of –1500-bp untranslated region to p < 0.02 versus the vector-transfected control, indicating that induction-fold decreased in proportion to the 5' deletion extent in the 5-untranslated region of the Bak promoter (Fig. 6A). To study the mechanism of G{alpha}s to inhibit hydrogen peroxide-induced Bak transcription mediated by transcription factors, the effects of G{alpha}s on the binding of AP1, CREB, NFAT, and NF-{kappa}B to the Bak promoters were analyzed by electrophoretic mobility shift assay in SH-SY5Y cells. The expression of G{alpha}sQL inhibited the hydrogen peroxide-induced binding of AP1 probe from 3.2 ± 4.3- to 1.6 ± 2.3-fold (p < 0.05), NFAT probe from 2.2 ± 4.1- to 1.7 ± 1.8-fold (p < 0.05), and NF-{kappa}B probe from 2.6 ± 4.4- to 1.7 ± 2.9-fold (p < 0.05) to the Bak promoter. However, G{alpha}sQL increased the basal binding of the CREB probe (2.2 ± 3.0-fold, p < 0.05) and inhibited the hydrogen peroxide-induced decrease in CREB binding from 0.17 ± 1.3- to 2.1 ± 2.9-fold (p < 0.05) (Fig. 6B). To assess the contribution of AP1 in Bak induction by hydrogen peroxide, the effect of two deletions in the AP1 site (–1221 to –1216 or –919 to –912) of (–1500)Bak-pLuc were analyzed. The deletions decreased hydrogen peroxide-induced luciferase activity from 6.1 ± 0.1-(p < 0.02) to 2.6 ± 0.5-fold, and to 1.8 ± 0.2-fold (p < 0.02), respectively, and deletion of both sites decreased to 1.1 ± 0.1-fold (p < 0.02) (Fig. 6C).


Figure 3
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  		FIGURE 3.
G{alpha}s inhibited the hydrogen peroxide-induced transcription of Bak in SH-SY5Y cells. A, effects of G{alpha}sQL on the degradation rate of Bak protein. SH-SY5Y cells were treated with 10 µg/ml cycloheximide (CHX), and Bak protein levels were quantified at various times by Western blotting analysis of total cell lysates. The graph shows the average densities of Bak bands. B and C, effects of G{alpha}sQL on Bak mRNA levels and Bak-luciferase activity. Bak mRNA expressions were measured by RT-PCR (B) and quantitative real-time RT-PCR (C). Amounts of Bak mRNA were normalized versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and presented as ratio versus vector transfected controls. For luciferase assay, SH-SY5Y cells were transfected with 2.5 µg of (–3500 bp)Bak-pLuc and 2.5 µg of a β-galactosidase construct. After 24 h, cells were treated with 100 µM hydrogen peroxide for 24 h, and luciferase activities were measured and normalized versus β-galactosidase activity and are presented as ratio versus vector-transfected controls. Asterisks indicate significant differences (p < 0.05 versus vector-transfected cells, Mann-Whitney U test).

 
G{alpha}s Inhibited Hydrogen Peroxide-induced Activation of NF-{kappa}B and NFAT in a PKA-dependent Pathway—To probe the mechanism for G{alpha}s-cAMP signaling to inhibit hydrogen peroxide-induced AP1 activation, the effects of G{alpha}s on the phosphorylation and expression of AP1 components: c-Jun and c-Fos. Treatment with hydrogen peroxide increased phosphorylation of c-Jun at Ser63 and Ser73, and expression of c-Jun and c-Fos. G{alpha}sQL expression blocked the hydrogen peroxide-induced phosphorylation of c-Jun, and inhibited the induction of c-Jun to 40.0 ± 7.9% (p < 0.02) and c-Fos to 60.0 ± 8.1% (p < 0.02) of that of the vector-transfected control (Fig. 6D). The phosphorylation of JNK, a major kinase that activates c-Jun was increased by hydrogen peroxide treatment. The expression of G{alpha}sQL also increased the basal phosphorylation of JNK as high as that of hydrogen peroxide-treated vector-transfected cells, without resulting in an increase in the phosphorylation and expression of c-Jun and c-Fos (Fig. 6D). Treatment of G{alpha}sQL-expressing cells with hydrogen peroxide did not cause a significant change in JNK phosphorylation. The expression of G{alpha}sQL also increased the phosphorylation of p38 and ERK mitogen-activated protein kinase as high as that of hydrogen peroxide-treated vector-transfected cells (data not shown), suggesting that G{alpha}s inhibits hydrogen peroxide-induced AP1 activation by inhibiting other enzymes. Thus, the involvement of glycogen synthase kinase-3β (GSK-3β) was examined, because GSK-3β was reported to involve in c-Jun induction. Treatment of SH-SY5Y cells with hydrogen peroxide decreased the phosphorylation of GSK-3β at serine 9 to activate the enzyme (Fig. 6E). G{alpha}sQL expression increased the basal phosphorylation level of GSK-3β (Ser9) (2.3 ± 0.1-fold, p < 0.05) and decreased the basal c-Jun level (0.8 ± 0.1-fold, p < 0.05) versus vector-transfected cells. G{alpha}sQL expression repressed the decrease in GSK-3β (Ser9) phosphorylation induced by hydrogen peroxide (2.1 ± 0.1- from 0.5 ± 0.2-fold in vector-transfected cells, p < 0.05) and in expression of c-Jun (0.8 ± 0.1-fold from 1.9 ± 0.1-fold, p < 0.05) (Fig. 6E). Treatment with lithium chloride (selectively inhibit GSK-3) increased the phosphorylation of GSK-3β, and decreased hydrogen peroxide-induced phosphorylation and induction of c-Jun in both vector- and G{alpha}sQL-transfected cells. Moreover, treatment with H89 also blocked the increase in GSK-3β phosphorylation (Ser9) induced by G{alpha}sQL (data not shown). This result suggests that G{alpha}s repress hydrogen peroxide-induced AP1 activation by inhibition of GSK-3β in a PKA-dependent pathway.

Next, the effects of G{alpha}s on the degradation of I{kappa}B{alpha} following hydrogen peroxide treatment were examined to probe the mechanism for G{alpha}s to inhibit hydrogen peroxide-induced activation of NF-{kappa}B. Treatment of SH-SY5Y cells with hydrogen peroxide decreased the I{kappa}B{alpha} level to activate NF-{kappa}B. G{alpha}sQL expression increased the basal expression level of I{kappa}B{alpha} versus vector-transfected cells (1.9 ± 0.1-fold, p < 0.05), and repressed the hydrogen peroxide-induced decrease in I{kappa}B{alpha} levels from 0.4 ± 0.1- to 1.7 ± 0.1-fold (p < 0.05) (Fig. 6F). Treatment with H89 slightly decreased the basal level of I{kappa}B{alpha}, but augmented the hydrogen peroxide-induced decrease in the I{kappa}B{alpha} level, suggesting G{alpha}s inhibit hydrogen peroxide-induced activation of NF-{kappa}B by PKA-dependent blocking of the I{kappa}B{alpha} degradation in SH-SY5Y cells. Finally, to probe the mechanism for G{alpha}s-cAMP signaling to inhibit hydrogen peroxide-induced NFAT activation, the effects of G{alpha}s on the nuclear translocation of NFAT following hydrogen peroxide treatment was analyzed, because NFATs are known to translocate into the nucleus after activation. Most of NFAT was localized in the cytosol fraction in SH-SY5Y cells before treatment, but hydrogen peroxide treatment induced translocation of most of NFAT from the cytosol to nuclear fraction (Fig. 6F). However, G{alpha}sQL expression decreased the hydrogen peroxide-induced translocation of NFAT to the nuclear fraction (0.2 ± 0.1- from 0.8 ± 0.2-fold, p < 0.05), without significant change in its distribution before the treatment. Treatment with H89 caused a decrease in cytosolic distribution and concomitant increase in nuclear distribution of NFAT in vector-transfected cells and G{alpha}s-transfected cells, suggesting that G{alpha}s inhibits hydrogen peroxide-induced NFAT activation by blocking its nuclear translocation in a PKA-dependent pathway.


Figure 4
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  		FIGURE 4.
G{alpha}s inhibited hydrogen peroxide-induced apoptosis of SH-SY5Y cells by engaging a cAMP-PKA-CREB pathway. A, effects of forskolin on the hydrogen peroxide-induced apoptosis and expression of Bcl2 family proteins. SH-SY5Y cells were treated with forskolin (15 µM) and hydrogen peroxide (100 µM) for 24 h. Caspase-3 and PARP cleavage, cytochrome c release, and Bcl2 family protein expressions were analyzed by Western blotting. B and C, effects of H89 or CRE-decoy on the anti-apoptotic effect of G{alpha}s. SH-SY5Y cells were pretreated with H89 (10 µM) for 30 min or transfected with CRE-decoy oligonucleotide (150 nM), and then treated with hydrogen peroxide for 24 h. Apoptosis was analyzed by Western blotting, and Bak mRNA expression by RT-PCR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 


Figure 5
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  		FIGURE 5.
G{alpha}s inhibited the activations of transcription factors induced by hydrogen peroxide in SH-SY5Y cells. A, effects of G{alpha}s and forskolin on hydrogen peroxide-induced activation of transcription factors. SH-SY5Y cells were transfected with CREB-, AP1-, NF-AT-, or NF-{kappa}B-luciferase constructs (2.5 µg) and β-galactosidase plasmid. After 24 h, cells were treated with hydrogen peroxide (100 µM) for 24 h, and then luciferase activities were analyzed. B, effects of transcription factor inhibitors on Bak transcription. SH-SY5Y cells were pretreated with SB203580 (10 µM), cyclosporine A (1 µM), or pyrrolidine dithiocarbamate (PDTC) (10 µM) for 30 min or transfected with CRE decoy (150 nM) and then treated with hydrogen peroxide for 24 h before analysis of the Bak luciferase activity. V represents vector-transfected cells and G represents G{alpha}sQL-transfected cells. Asterisks indicate significantly different from the control cells (p < 0.05, Mann-Whitney U test).

 


Figure 6
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  		FIGURE 6.
G{alpha}s repressed hydrogen peroxide-induced Bak expression by inhibiting the binding of transcription factors on Bak promoter in a PKA-dependent manner. A, effects of serial deletions in the Bak promoter on Bak-luciferase activity induced by hydrogen peroxide. B, effects of G{alpha}sQL on the binding of AP1, CREB, NFAT, and NF-{kappa}B to Bak promoter. The bindings of transcription factors to Bak promoter were analyzed by electrophoretic mobility shift assay using a chemiluminescent biotin 5' end-labeled DNA probe corresponding to the AP1, CREB, NFAT, or NF-{kappa}B consensus sequences. For competition experiments, 100-fold excesses of unlabeled AP1, CREB, NFAT, or NF-{kappa}B oligonucleotide were added. C, effects of AP1 binding site deletions on Bak-luciferase activity induced by hydrogen peroxide. SH-SY5Y cells were co-transfected with Bak-luciferase plasmids containing various deletions or AP1 binding site deletion in Bak promoter and β-galactosidase plasmids, and then Bak luciferase activity was analyzed and normalized to β-galactosidase. D, effects of G{alpha}s on the hydrogen peroxide-induced activation of JNK. E, effects of G{alpha}s on the hydrogen peroxide-induced activation of GSK-3β. F, effects of G{alpha}s on the hydrogen peroxide-induced degradation of I{kappa}B{alpha} and nuclear translocation of NFAT. SH-SY5Y cells expressing G{alpha}sQL were treated with hydrogen peroxide (100 µM) for 12 h in the presence or absence of 10 mM LiCl or 10 µM H89. The expressions and phosphorylations of JNK, GSK-3β, NFAT, c-Jun, and c-Fos were analyzed by Western blotting using a respective specific antibody. For analysis of nuclear translocation of NFAT, cytosolic and nuclear proteins were prepared. Histograms represent mean ± S.E. of band densities expressed as -fold of the corresponding densities of control cells.

 


Figure 7
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  		FIGURE 7.
GPCR ligands regulated the hydrogen peroxide-induced apoptosis of SH-SY5Y cells. A, effects of PGE2 on hydrogen peroxide-induced apoptosis. SH-SY5Y cells were pretreated with 10 µM PGE2 for 30 min before hydrogen peroxide treatment. B, effects of PGE2 and forskolin on the temporal patterns of cAMP and CREB phosphorylation. SH-SY5Y cells were treated with 10 µM PGE2 (circle) or 15 µM forskolin (triangle), and then cAMP concentration (solid lines) and CREB phosphorylation (dotted lines) were analyzed at the indicated times. cAMP levels were analyzed by measuring competitive binding of [3H]cAMP to the regulatory subunit of cAMP-dependent protein kinase. CREB phosphorylation was analyzed by Western blotting using a phosphorylated CREB (Ser-133)-specific antibody. The blots shown are representative of at least three independent experiments, and the graph represent average values (mean ± S.E.) at the time intervals. C, effects of G{alpha}i2QL on hydrogen peroxide-induced apoptosis. SH-SY5Y cells were transfected with 15 µg of pcDNA3-G{alpha}i2QL plasmid and then treated with hydrogen peroxide (100 µM) for 24 h. D, effects of CCPA on hydrogen peroxide-induced apoptosis. SH-SY5Y cells were pretreated with 10 µM CCPA (a selective A1R agonist) for 30 min before being treated with hydrogen peroxide (100 µM) for 24 h. Data are expressed as -fold of corresponding densities of caspase-3 (empty bar) and PARP cleavages (filled bar) to that of the control cells, and the histograms represent mean ± S.E. Asterisks indicate significant differences (p < 0.05 versus control cells, Mann-Whitney U test).

 
GPCR Ligands Modulated the Hydrogen Peroxide-induced Apoptosis of SH-SY5Y Neuroblastoma Cells—Because G{alpha}s was found to inhibit the hydrogen peroxide-induced apoptosis of SH-SY5Y neuroblastoma cells, we examined the effects of PGE2, which binds to the G{alpha}s-coupled receptor to stimulate adenylate cyclases, on hydrogen peroxide-induced Bak expression and apoptosis. Treatment with PGE2 reduced hydrogen peroxide-induced CREB dephosphorylation at Ser133, Bak expression, and the cleavages of PARP and caspase-3. The cleavage of caspase-3 was also decreased by PGE2 from 5.5 ± 0.4- to 2.7 ± 0.7-fold (p < 0.02), and that of PARP was from 6.4 ± 0.5- to 3.7 ± 0.3-fold (p < 0.05) (Fig. 7A). Because PGE showed anti-apoptotic effects similar to the forskolin effect, we examined the temporal patterns of cAMP concentration and CREB phosphorylation at serine 133 following PGE2 and forskolin treatments. Treatment of SH-SY5Y cells with forskolin resulted in a rapid increase in cAMP concentration that reached a peak at 30 min (110.4 ± 3.2 pmol/mg of protein), and CREB phosphorylation that reached a peak at 3 h (38.9 ± 2.5-fold). Both the elevated cAMP level and CREB phosphorylation by forskolin decreased so slowly that they remained at elevated levels until 24 h after treatment (31.6 ± 4.6 pmol/mg protein and 15.5 ± 3.7-fold, respectively). In contrast, PGE2 treatment caused a rapid increase in the cAMP level with a peak at 30 min (78.3 ± 3.9 pmol/mg of protein), a sharp decrease to about half the peak value at 1 h, and then a slow decrease to reach the basal level at 6 h after PGE2 treatment. In contrast to the cAMP level, CREB phosphorylation induced by PGE2 reached a peak (31.6 ± 4.2-fold) at 1 h, and then slowly decreased to maintain elevated levels of phosphorylation (10.8 ± 4.3-fold p < 0.05) until 24 h, showing a similar temporal pattern of CREB phosphorylation by forskolin. This result suggests that PGE2 can maintain CREB activation for several hours after cAMP concentration is elevated for a short period in SH-SY5Y cells (Fig. 7B).

Furthermore, the expression of constitutively active G{alpha}i2 (G{alpha}i2QL), which antagonizes G{alpha}s effects, augmented hydrogen peroxide-induced Bak expression and the cleavages of caspase-3 and PARP, and partially eliminated the inhibitory effect of G{alpha}sQL on hydrogen peroxide-induced Bak expression and cleavages of caspase-3 and PARP (Fig. 7C). However, G{alpha}i2QL expression also increased the basal expression level of the Bak protein in vector-transfected cells and G{alpha}sQL-transfected cells, implying that G{alpha}i2 may increase basal Bak expression by a CREB-independent pathway because G{alpha}i2 was shown to decrease CREB phosphorylation in SH-SY5Y cells (data not shown). Treatment with CCPA, which (binding of CCPA to adenosine A1 receptor activates G{alpha}i2 to inhibit adenylate cyclases) augments hydrogen peroxide-induced Bak expression, CREB dephosphorylation, and cleavages of PARP and caspase-3. The hydrogen peroxide-induced cleavage of caspase-3 was increased by CCPA from 5.8 ± 0.3- to 7.9 ± 0.7-fold (p < 0.02), and of PARP from 6.8 ± 0.1- to 9.9 ± 0.1-fold (p < 0.05) (Fig. 7D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study was performed to determine whether the {alpha} subunit of stimulatory G protein, G{alpha}s, can modulate hydrogen peroxide-induced apoptosis and if so, to elucidate the molecular mechanism responsible. We found that G{alpha}s inhibits hydrogen peroxide-induced apoptosis by repressing Bak induction in SH-SY5Y human neuroblastoma cells. This finding is supported by the following results. First, the overexpression of constitutively active G{alpha}sQL was found to protect SH-SY5Y cells from hydrogen peroxide-induced apoptosis, and G{alpha}s siRNA augmented apoptosis. Second, G{alpha}sQL overexpression repressed the hydrogen peroxide-induced up-regulation of Bak protein by inhibiting transcription of Bak. Third, Bak overexpression abolished the protective effect of G{alpha}s on hydrogen peroxide-induced apoptosis, and knockdown of Bak (using siRNA) inhibited the apoptosis. We also found that the repression of Bak induction by G{alpha}s is mediated by cAMP, PKA, and CREB. This was evidenced by the observations that Bt2cAMP and forskolin repressed Bak induction and protected cells against hydrogen peroxide-induced apoptosis; moreover, treatment with the PKA inhibitor H89 or transfection with decoy CRE restored Bak promoter activity, augmented hydrogen peroxide-induced apoptosis, and abolished the protective effect of G{alpha}sQL. In addition, the protective effect of G{alpha}s was abolished by co-expressing constitutively active mutant G{alpha}i2, which antagonizes G{alpha}s by inhibiting adenylate cyclase.

The G{alpha}s-cAMP signaling pathway has been reported to regulate cell death in various ways. Treatment with cholera toxin (which activates G{alpha}s) was found to protect neuronal cells from glutamate-induced ROS-mediated apoptosis by up-regulating the anti-apoptotic protein Bcl2 in a cAMP-dependent manner (15), and forskolin has been reported to prevent the hydrogen peroxide-induced apoptosis of PC12 pheochromocytoma cells by increasing the level of antioxidant glutathione (21). Bt2cAMP inhibited apoptosis by blocking the induction of Bax mRNA (22). Moreover, PKA was reported to protect cells from apoptosis by inducing Bad phosphorylation (23), whereas CREB induces the transcription of the anti-apoptotic gene Bcl2. However, it has not been previously reported that the G{alpha}s-cAMP signaling pathway modulates apoptosis by regulating Bak expression. Thus, this study presents the first evidence, to the best of our knowledge, that the G{alpha}s-cAMP signaling pathway modulates cancer cell apoptosis by repressing Bak induction.

Bak protein is a multidomain pro-apoptotic protein of the Bcl2 family, and acts as an essential gateway to intrinsic cell death pathways by facilitating the release of cytochrome c from mitochondria (24, 25). Moreover, Bax–/–Bak–/– double knock-out cells have been reported to be resistant to anticancer drug-induced apoptosis in many human cancer cells (26). In addition, Bak has been reported to be up-regulated and to act as a downstream mediator of an oxidative stress pathway that leads to the apoptosis of SH-SY5Y neuroblastoma cells in response to fenretinide (27). These reports demonstrate that Bak mediates hydrogen peroxide-induced apoptosis, and support our finding that G{alpha}s can exert a protective effect against hydrogen peroxide-induced apoptosis by repressing Bak expression in SH-SY5Y neuroblastoma cells.

In our study to investigate the mechanism underlying the repression of hydrogen peroxide-induced Bak expression by G{alpha}s, we found that G{alpha}s represses Bak expression by inhibition of activation and binding of several transcription factors, i.e. AP1, NF-{kappa}B, and NFAT, to Bak promoter following hydrogen peroxide treatment. This conclusion was reached because the transcription factors AP1, NFAT, and NF-{kappa}B, but not CREB, were found to be activated by hydrogen peroxide when assessed by luciferase assay; specific inhibitors of the transcription factors blocked Bak transcription induced by hydrogen peroxide; and G{alpha}s or forskolin were found to inhibit the hydrogen peroxide-induced activations of AP1, NFAT, and NF-{kappa}B, and thus to repress Bak gene transcription. In addition, G{alpha}s also inhibited the binding of AP1, NF-{kappa}B, and NFAT to the Bak promoter induced by hydrogen peroxide treatment. The binding sites for these transcription factors seems to be scattered widely in the Bak promoter region (–3500 bp), because Bak luciferase was found to be increased in proportion to the extent of deletion in this region. The nuclear transcription factor AP1 is composed of dimers of fos and jun proto-oncogenes, and has been linked to a variety of cellular events including proliferation, differentiation, transformation, and apoptosis. Moreover, oxidative stress is known to induce the expressions and phosphorylations of Fos and Jun (28, 29), and the DNA binding activity of AP1 in various cell types is known as a prelude to cell death (30, 31). This study indicates that AP1 is involved in the repression of hydrogen peroxide-induced Bak expression by G{alpha}s. This is based on the results that the deletion of the two AP1 binding sites in the Bak promoter blocked Bak induction by hydrogen peroxide, and that G{alpha}s repressed the expression and phosphorylation of both c-Fos and c-Jun. Recently, it was reported that nitric oxide activates an apoptotic cascade in human brain tumor cells, involving sustained JNK activation to activate c-Jun and AP1 DNA binding activity and subsequent Bak induction (32). These reports show that AP1 mediates the apoptosis induced by oxidative stress, and support our finding that the repression of AP1 activation by the G{alpha}s-cAMP signaling system protects cells from apoptosis. We found that G{alpha}s represses hydrogen peroxide-induced activation of GSK-3β by phosphorylation of GSK-3β at serine 9, which can result in inhibition of the phosphorylation and induction of c-Jun to repress AP1 activation. Furthermore, a GSK-3 inhibitor, lithium, was found to block the hydrogen peroxide-induced phosphorylation and induction of c-Jun. The phosphorylation of GSK-3β in G{alpha}sQL-expressing cells were blocked by the PKA inhibitor, as suggested by the report that PKA can directly phosphorylate GSK-3β at serine 9 and inhibit its apoptotic activity in neurons (33). GSK-3 was reported to act in tandem with JNK to coordinate the full execution of the c-Jun stress response and AP-1-induced death (34). Together with these reports, our finding suggests that G{alpha}s-cAMP signaling inhibits hydrogen peroxide-induced AP1 activation by PKA-dependent phosphorylation and inhibition of GSK-3β. However, G{alpha}sQL expression increased the basal and hydrogen peroxide-stimulated phosphorylation of JNK, p38, and ERK without a significant increase in the phosphorylation and expression of c-Jun and c-Fos, suggesting that G{alpha}s repressed hydrogen peroxide-induced AP1 activation in a mechanism that is independent of JNK, p38, and ERK. NF-{kappa}B-induced transcription has been reported to play a central role in host defense and inflammatory response, and to prevent apoptosis by increasing the expressions of anti-apoptotic genes including Bcl2, and by decreasing the expression of pro-apoptotic Bax protein in many human tumors. Thus, NF-{kappa}B may be a major factor that controls the ability of cells to resist apoptosis-based tumor surveillance systems (35). However, NF-{kappa}B has also been reported to stimulate apoptosis by inducing the expressions of death-promoting genes including p53, the death receptor Fas, and its ligand, TNF-{alpha}, and Bak (36–39) and by linking fenretinide-induced ROS production to apoptosis. NF-{kappa}B dimers are held in an inactive cytoplasmic complex with a family of inhibitory proteins, the I{kappa}Bs, including I{kappa}B{alpha}. Phosphorylation of I{kappa}Bs at two serine residues in their N-terminal regulatory domain by the I{kappa}B kinase complex targets them for rapid ubiquitin-mediated proteasomal degradation, which induces the nuclear translocation of NF-{kappa}Bto function (40). Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide were reported to inhibit the activation-induced cell death in T cells by repressing expression of Fas ligand through stabilization of I{kappa}B{alpha} via the cAMP signaling system (41). cAMP was found to decrease NF-{kappa}B activation in response to the superoxide anion (42, 43), and to increase the I{kappa}B{alpha} levels to inhibit lipopolysaccharide-induced NF-{kappa}B activation in THP-1 cells (44). Furthermore, activation of the PKA pathway is suggested to inhibit NF-{kappa}B transcription by phosphorylating CREB, which competes with p65 for limiting amounts of CBP, because both NF-{kappa}B and NFAT require the coactivator CBP for transcriptional activity (45). These reports support our finding that G{alpha}s increases the I{kappa}B{alpha} level in a PKA-dependent pathway to inhibit hydrogen peroxide-induced activation of NF-{kappa}B in neuroblastoma cells. This finding indicates that the G{alpha}s-cAMP signaling pathway protects cells from apoptosis by inhibiting the hydrogen peroxide-induced activation of NF-{kappa}B, and implies that NF-{kappa}B mediates apoptosis by stimulating Bak expression. NFATs constitute a family of transcription factors that transduce calcium signals and coordinate the expressions of proteins involved in cell growth and apoptosis. NFAT is known to be activated by activation of various receptors inducing calcium mobilization, and the receptor activation and calcium mobilization result in activation of many intracellular enzymes including the calcium- and calmodulin-dependent phosphatase calcineurin. Calcineurin is a major upstream regulator of NFAT proteins, and causes the dephosphorylation and translocation of NFAT proteins from the cytoplasm to the nucleus of activated cells (46, 47). NFAT has been reported to mediate oxidative stress-induced apoptosis by inducing Fas ligand expression. Moreover, Bcl2 and Bcl-xL were found to mediate anti-apoptotic effects by inhibiting NFAT activation and translocation to the nucleus through the sequestration of calcineurin (48). cAMP signaling has been reported to inhibit NFAT activity by direct phosphorylation of NFAT by PKA resulting in inhibition of nuclear entry and transcriptional activity (41, 49). Together with these reports, our finding that G{alpha}s inhibits hydrogen peroxide-induced nuclear translocation of NFAT, which was inhibited by a PKA inhibitor, suggest that G{alpha}s-cAMP signaling inhibits hydrogen peroxide-induced activation of NFAT by a PKA-dependent phosphorylation. This study shows that the G{alpha}s-cAMP signaling system might act upstream of transcription factors such as AP1, NF-{kappa}B, and NFAT to block their activation induced by hydrogen peroxide, the resulting Bak expression, and then apoptosis of SH-SY5Y neuroblastoma cells. The present study also shows that G{alpha}sQL expression increases the basal level of the Bak protein and CREB binding to the Bak promoter, but that G{alpha}sQL expression inhibits Bak induction by hydrogen peroxide. Furthermore, it was also shown that hydrogen peroxide reduced CREB binding to the Bak promoter, and that the transfection of decoy CREB increased hydrogen peroxide-induced Bak luciferase activity. Thus, to interpret this seemingly paradoxical phenomenon, we speculate that the G{alpha}s-cAMP signaling system stimulates basal CREB binding to the Bak promoter to increase the basal level of transcription, which is reduced by hydrogen peroxide, but that it represses hydrogen peroxide-induced Bak expression by inhibiting activation of transcription factors mediating Bak expression in a CREB-dependent manner. In addition to Bak expression, G{alpha}s was found to represses the hydrogen peroxide-induced down-regulation of Bcl-xL in SH-SY5Y human neuroblastoma cells, and the resulting increase in Bcl-xL expression shifted the ratio of anti- to pro-apoptotic Bcl2 family proteins to favor cell survival (50). This finding suggests that the G{alpha}s-cAMP signaling system maintains mitochondrial integrity and protects cells from apoptosis not only by repressing the induction of pro-apoptotic Bak but also by maintaining the levels of anti-apoptotic Bcl-xL proteins, which implies that G{alpha}s-cAMP affects multiple pro- and anti-apoptotic Bcl2 family proteins.

Because G{alpha}s was found to protect cells from hydrogen peroxide-induced apoptosis, we examined whether PGE2, anagonist of the receptors that couple with G{alpha}s, protects SH-SY5Y cells from hydrogen peroxide-induced apoptosis, and found this to be the case. This finding is similar to reports that PGE2 protects human colon cancer cells from apoptosis (51, 52). In fact, EP receptors have been identified as potential targets for the treatment and prevention of colorectal cancer (53). PGE2 binds to four different receptors termed EP1, EP2, EP3, and EP4, and of these EP2 and EP4 couple to G{alpha}s (54). PGE2 induced a transient increase in cAMP levels, but maintained CREB phosphorylation for several hours, which might be involved in the anti-apoptotic effect of the PGE2-G{alpha}s signaling system. On the other hand, treatment with CCPA, an adenosine A1 receptor agonist, enhanced hydrogen peroxide-induced apoptosis in SH-SY5Y neuroblastoma cells. Adenosine A1 receptor interacts with the pertussis toxin-sensitive Gi protein that inhibits adenylate cyclase, and is considered a promising therapeutic target in cancer (55). The anti-apoptotic effect of PGE2 and the pro-apoptotic effect of CCPA were accompanied by corresponding changes in Bak induction after hydrogen peroxide treatment, indicating that both PGE2 and CCPA influence apoptosis by modulating the G{alpha}s/G{alpha}i-cAMP signaling pathway that regulates Bak expression. This result suggests that apoptosis induced by various stimuli including {gamma}-radiation and chemotherapeutic agents, thus their therapeutic efficiency, can be improved by co-treatment with specific GPCR ligands that modulate sensitivity or resistance to apoptosis.

Based on the findings of the present study, we concluded that G{alpha}s can protect neuroblastoma cells from hydrogen peroxide-induced apoptosis by repressing Bak induction, and that this is mediated by the inhibition of hydrogen peroxide-induced transcription factor activation (e.g. AP1, NF-{kappa}B, and NFAT) by the cAMP-PKA-CREB signaling pathway. We also conclude that hydrogen peroxide-induced apoptosis can be modulated by the administration of receptor agonists that activate G{alpha}s or G{alpha}i. These findings provide a better understanding of how G{alpha}s signaling pathways modulate ROS-induced apoptosis, and have important clinical implications, namely, that the apoptosis of cancer cells mediated by hydrogen peroxide following chemotherapy or radiation can be enhanced by administering agonists or antagonists of cancer cell-specific receptors to improve the therapeutic efficiency.


   FOOTNOTES
 
* This work was supported by Grants M2-0408-00-0027 and M2-0513-00-0099 from the Nuclear R&D Program of the Ministry of Science & Technology and the 2007 BK21 Project for Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Jongno-gu, Seoul 110-799, Republic of Korea. Tel.: 82-2-740-8247; Fax: 82-2-744-4534; E-mail: juhnn{at}snu.ac.kr.

2 The abbreviations used are: G-protein, GTP-binding protein; CCPA, 2-chloro-N(6)-cyclopentyl-adenosine; CREB, cyclic AMP response element-binding protein; G{alpha}s, {alpha} subunit of stimulatory G proteins; GPCR, G protein-coupled receptor; GSK-3, glycogen synthase kinase-3; JNK, c-Jun N-terminal kinase; PGE2, prostaglandin E2; PKA, cAMP-dependent protein kinase; ROS, reactive oxygen species; Bt2cAMP, dibutyryl cyclic AMP; FACS, fluorescence-activated cell sorter; siRNA, small interfering RNA; RT, reverse transcriptase; PARP, poly(ADP-ribose) polymerase; ERK, extracellular signal-regulated kinase; EP, prostaglandin E receptor. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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